Using Salivary Glands as a Tissue Target for Gene Therapeutics

Gene transfer offers a potential way to correct local and systemic protein deficiency disorders by using genes as drugs, so called gene therapeutics. Salivary glands present an interesting target site for gene therapeutic applications. Herein, we review proofs of concept achieved for salivary glands with in vivo animal models. In that context we discuss problems (general and salivary tissue-specific) that limit immediate clinical use for this application of gene transfer. Ongoing efforts, however, suggest that salivary glands may be suitable as gene therapeutic target sites for drug delivery in the near future.     

Prospects for gene-based immunopharmacology in salivary glands

The clinical potential of gene transfer is increasing. One likely major application of this emerging biotechnology will be for gene therapeutics, the use of a gene as a drug. Salivary glands provide an unusual but increasingly valuable target site for gene transfer. Studies in animal salivary glands from several laboratories, including our own, have provided proof of this concept. In this review, we provide a background and perspective on possible strategies for gene-based immunopharmacology in salivary glands. We use as a target disease model the autoimmune exocrinopathy Sj?gren's syndrome.     

CP‐25 Alleviates Experimental Sjögren's Syndrome Features in NOD/Ltj Mice and Modulates T Lymphocyte Subsets

Primary Sjögren's syndrome (pSS) is a chronic inflammatory autoimmune illness of the moisture‐producing glands such as salivary glands that is characterized by various immune abnormalities. The aetiology of pSS remains unclear and there is no curative agent. In this study, we investigated the putative therapeutic effects on a NOD/Ltj mouse model of Sjögren's syndrome‐like disorders of an ester derivative of paeoniflorin, paeoniflorin‐6′O‐benzene (termed CP‐25). Our study showed that CP‐25 alleviated effectively clinical manifestations in NOD/Ltj mice resulting, for example, in increased salivary flow and reduced histopathological scores. Furthermore, CP‐25 decreased lymphocyte viability in NOD/Ltj mice and attenuated the infiltration of Th1 cells and Th2 cells into the salivary glands of NOD/Ltj mice. In the spleen on NOD/Ltj mice, CP‐25 skewed the ratio of Th17 and regulatory T cells towards regulatory T cells. After treatment, concentrations of anti‐La/SSB and IgG antibodies were reduced and the titre of the inflammatory cytokines IFN‐γ, IL‐4, IL‐6 and IL‐17A in the serum on NOD/Ltj mice was alleviated. Thus, we define CP‐25 as a novel compound that is a potent therapeutic agent for pSS by modulating T lymphocyte subsets. Future studies will validate the use of CP‐25 as a therapeutic strategy for the treatment of pSS.     

Alleviating effect of paeoniflorin-6′-O-benzene sulfonate in antigen-induced experimental Sjögren’s syndrome by modulating B lymphocyte migration via CXCR5-GRK2-ERK/p38 signaling pathway

Primary Sjögren's syndrome (pSS) is an autoimmune disease of unresolved aetiology that affects the exocrine glands. Clinical symptoms frequently also involve skin, liver, kidney and neurovascular components. The pathogenesis of pSS is still unclear but B cell hyperactivity has been identified as a hallmark of pSS. Currently, a curative therapeutic agent is lacking. In this study, we explored whether paeoniflorin-6′-O-benzene (CP-25) exerted therapeutic effects through regulating B lymphocyte migration via CXCR5-GRK2-MAPK mediated signaling pathways in a mouse model of antigen-induced, experimental Sjögren's syndrome (ESS). We found that CP-25 increased the salivary flow and alleviated the histopathology of ESS. Furthermore, CP-25 reduced the viability of B lymphocyte and limited the target organs index. In the peripheral blood and salivary gland of ESS mice, CP-25 down-regulated the proportion of total B cells, CXCR5+ B cells and PDCA1 + CD19- and limited the presence of phosphorylated (p-) p38 and ERK (p-ERK). Besides, CP-25 increased the percentage of memory B cells in the peripheral blood and reduced it in salivary gland. Furthermore, in vitro, CP-25 down-regulated p-p38, p-ERK, CXCR5 and membrane GRK2, and increased cytoplasm GRK2 in Maver-1 cells, a mantle cell lymphoma cell line, causing a lower migration ability of Maver-1 cells. Thus, we define CP-25 as a novel compound that is a potent therapeutic agent for pSS which modulates B lymphocyte subsets and impacts the migration of B lymphocytes through regulating the CXCR5-GRK2-ERK/p38 signaling pathway.     

104例涎腺肿瘤的临床分析

通过分析104例涎腺肿瘤的临床表现、诊断及治疗,发现男女患者的比值为1:1.21;良性肿瘤80例,占76.9％;恶性肿瘤24例,占23.0％;良性肿瘤多见于21～50岁组(63.25％),主要为混合瘤、腺淋巴瘤;恶性肿瘤多见于51～60岁组(58.33％),主要为粘液表皮样癌、恶性混合瘤、腺样囊性癌;肿瘤的好发部位依次为:腮腺部、颌下腺部、小涎腺部及舌下腺部。其中腮腺及颌下腺部良性肿瘤高于恶性肿瘤;小涎腺中的恶性肿瘤比重大大增加。诊断中强调了细针吸细胞学检查的价值,并对肿瘤的手术原则及预后作了讨论。     

Imidacloprid-mediated alterations on the salivary glands of the Neotropical brown stink bug,Euschistus heros

The management of the Neotropical brown stinkbug Euschistus heros (Hemiptera: Pentatomidae) in soybean fields has been heavily dependent on the application of neonicotinoid insecticides. Neonicotinoids act primarily by impairing the function of the nicotinic acetylcholine receptors of the nervous system. These compounds also target specific organs (e.g., salivary glands), which may potentiate their insecticidal efficacy. Here, we evaluated whether the exposure to the neonicotinoid imidacloprid would cause cytomorphological changes in the salivary glands of E. heros. First, we determined the lethal concentrations (LCs) of imidacloprid through contact and ingestion. Subsequently, the cytomorphology of the salivary glands were evaluated in insect groups that survived exposure to the LC₅ (3.75 mg a.i./L), LC₅₀ (112.5 mg a.i./L), or LC₇₅ (375.0 mg a.i./L, equivalent to the recommended field rate) doses. Imidacloprid induced apoptosis and necrosis in the salivary gland cells according to the insecticide concentration and salivary gland region. All concentrations increased apoptosis and injured cells (e.g., vacuolization, chromatin condensation, swelling of organelles, and plasma membrane rupture) in the principal and accessory salivary glands. Individuals that survived exposure to the highest concentrations (i.e., LC₅ and LC₅₀) were more affected, and exhibited several necrotic cells on their main principal salivary glands. Collectively, our results indicate that imidacloprid exerts toxic effects on the non-target organs, such as the salivary glands, which increases the efficacy of this compound in the management of stink bug infestations.

     

Oxidative stress induced by destruxin from Metarhizium anisopliae (Metch.) involves changes in glutathione and ascorbate metabolism and instigates ultrastructural changes in the salivary glands of Spodoptera litura (Fab.) larvae.

The cyclodepsipeptidic mycotoxin, destruxin, produced by Metarhizium anisopliae is known for its larvicidal properties. The crude destruxin-treated Spodoptera litura larvae revealed a decrease in thiol content and an increment in oxidation of glutathione to glutathione disulfide and ascorbate to dehydro ascorbate in a dose- and time-dependent manner. An increase in the levels of protein carbonyls and the free radicals, hydrogen peroxide and hydroxyl radical was also observed. These provide a clear indication of oxidative stress. Upon ingestion, destruxin causes serious damage to the epithelial cells of the midgut. We report the ultrastructural effects of crude destruxin on the salivary glands of 9-day-old S. litura larva after 24 h of treatment with the mycotoxin at a dosage of 0.147 microg/g body wt. (LD(50)). The transmission electron microscopic observations revealed characteristic changes in the salivary glands of S. litura which include detachment and damage of the microvilli, epithelial cell vacuolization, bleeding of the epithelial cells into the salivary gland lumen and disruption of the epithelial cell membrane. This investigation focuses salivary glands also as the target organ of destruxin apart from the already known midgut, Malphigian tubules and haemocytes.     

Ion channel gene therapy for smooth muscle disorders: relaxing smooth muscles to treat erectile dysfunction

The promise of gene therapy to treat diseases remains largely unfulfilled. Past setbacks and the complexity of the delivery systems used, in terms of both targeting the appropriate cells and inducing expression of products at therapeutic levels, thus far have prevented significant success for gene therapy. Smooth muscle disorders represent a unique target for gene therapy. In many cases, smooth muscle is readily accessible and, to induce a therapeutic effect, will not require very high levels of gene product expression. This allows a lower efficiency of gene transfer to be successful. With these important features in mind, we believe that naked DNA transfer of potassium ion channels represents a novel and successful way to treat smooth muscle disorders. Herein, we present a rationale for treating erectile dysfunction, a smooth muscle disorder of the cavernosal bodies of the penis, with naked DNA gene transfer therapy. By inserting the hSlo gene, which codes for Maxipotassium channels, into smooth muscle cells, we can improve smooth muscle relaxation in the corporal bodies and thus improve erectile function. This method of gene transfer has proven to be safe and effective for erectile dysfunction, and human trials are ongoing.     

Salivary glands and noradrenergic transmission in diabetic rats

1 Type 2 diabetes is associated with diverse oral pathologies in which salivary flow reduction is one of the causes of these oral abnormalities. Scarce literature exists regarding noradrenergic transmission and adrenergic-induced salivary flow in submaxillary and parotid glands of type 2 diabetic rats. 2 We studied noradrenergic transmission as well as the secretory response to alpha1- and beta-adrenoceptor stimulation in the parotid and submaxillary glands of type 2 diabetic rats. 3 Diabetic rats exhibited diminished neuronal uptake, release and endogenous content of noradrenaline (NE) in both salivary glands. Further, NE synthesis was also diminished accompanied by decreased tyrosine hydroxylase activity. Salivary flow responses to alpha1-(methoxamine) and beta-(isoprenaline) adrenoceptor stimulation were reduced in the submaxillary as well as the parotid glands of diabetic rats. 4 Our results suggest that the reduction of noradrenergic transmission in the salivary glands of type 2 diabetic rats is in part responsible for the diminished salivary flow evoked by alpha1- and beta-adrenergic stimulation. Reduced noradrenergic activity may contribute to the pathophysiology of oral abnormalities in diabetic patients.     

Therapeutic drug monitoring in saliva. An update.

This article re-examines the issue of salivary therapeutic drug monitoring (STDM). The anatomy and physiology of saliva and the salivary glands, as well as the effects of disease and drugs on salivary secretion and composition, are discussed briefly. Drugs for which therapeutic drug monitoring (TDM) has been shown useful are individually considered to determine if salivary drug concentrations (Csal) are reflective of plasma free drug concentrations (C(up)). That is, is the Csal/C(up) ratio time- and concentration-independent, as supported by a review of literature data? The primary determinant which appears to govern the potential utility of STDM for many of the drugs is the pKa of the drug. Drugs which are not ionisable or are un-ionised within the salivary pH range (phenytoin, carbamazepine, theophylline) are candidates for STDM based on current literature data. Digoxin and cyclosporin are potential candidates for STDM; however, further studies are necessary to confirm these preliminary findings. On the basis of current literature data, STDM does not appear to be useful for other drugs therapeutically monitored in serum/plasma.     

Site directed vascular gene delivery in vivo by ultrasonic destruction of magnetic nanoparticle coated microbubbles

Site specific vascular gene delivery for therapeutic implications is favorable because of reduction of possible side effects. Yet this technology faces numerous hurdles that result in low transfection rates because of suboptimal delivery. Combining ultrasonic microbubble technology with magnetic nanoparticle enhanced gene transfer could make it possible to use the systemic vasculature as the route of application and to magnetically trap these compounds at the target of interest. In this study we show that magnetic nanoparticle-coated microbubbles bind plasmid DNA and successfully deliver it to endothelial cells in vitro and more importantly transport their cargo through the vascular system and specifically deliver it to the vascular wall in vivo at sites where microbubbles are retained by magnetic force and burst by local ultrasound application. This resulted in a significant enhancement in site specific gene delivery compared with the conventional microbubble technique. Thus, this technology may have promising therapeutic potential.From the Clinical EditorThis work focuses on combining ultrasonic microbubble technology with magnetic nanoparticle enhanced gene transfer to enable targeted gene delivery via the systemic vasculature and magnetic trapping of these compounds at the target of interest.     

Lipopolysaccharide-induced subsensitivity of protease-activated receptor-2 in the mouse salivary glands in vivo

Protease-activated receptor-2 (PAR-2) acts as a modulator of multiple physiological/pathophysiological functions including salivary exocrine secretion. Given the supersensitivity of endothelial PAR-2 under endotoxaemia, we investigated if endotoxin/lipopolysaccharide (LPS) could alter the sensitivity of PAR-2 in the salivary glands. The in vivo salivation in response to i.v. administration of the PAR-2-activating peptide SLIGRL-NH2, but not of carbachol, gradually decreased 6-20 h after LPS administration in the mice. The LPS-induced hyporeactivity to the PAR-2 agonist was partially reversed by repeated administration of aprotinin, a non-specific protease inhibitor. PAR-2 mRNA levels in the salivary glands, as assessed by the semi-quantitative RT-PCR analysis, remained unchanged following LPS challenge. Our findings indicate that in contrast to the supersensitivity of endothelial PAR-2 as described previously, subsensitivity of PAR-2 in the salivary glands develops during the LPS-induced systemic inflammation, which might involve desensitisation of PAR-2 by endogenous proteases.     

Segmental and intracellular distribution of lead in rat kidney and salivary glands

The segmental and intracellular distribution of lead (Pb) was studied in the kidney and salivary gland of Sprague-Dawley rats. Lead acetate was administered i.v. in a single dose (10 or 65 mg/kg body wt) or multiple biweekly doses (subchronic: 7 X 10 mg/kg over 3 months; chronic: 13 X 10 mg/kg over 6 months). Segments of cortical nephrons and salivary glands were separated following tissue slicing, incubation with collagenase and centrifugation on a Percoll density gradient medium. Subcellular fractions were obtained by differential centrifugation of renal and salivary tissue homogenates. Lead was predominantly localized in the renal proximal tubules, which contained at least twice as much of the metal as the distal tubules. Segment populations prepared from salivary tissues contained far less Pb than the renal fractions and showed no clear differences among themselves in their affinity for the metal. Intracellular Pb distribution was as follows: kidney nuclei (Nu) greater than mitochondria (Mt) greater than cytosol (Cy) greater than microsomes (Mc); salivary gland Cy greater than Mc greater than Mt greater than Nu. In most cases, 45Ca followed the same intracellular distribution as lead. Our data suggest that the proximal tubular segment may be the most likely renal target of chronic lead toxicity. The results point also to a much greater retention of Pb by the kidney than by salivary glands. The ability of the kidney to accumulate a great deal of lead to be released into tubular fluid over long periods, makes urinary lead a poor indicator of duration and frequency of exposure. On the other hand, the inability of salivary glands to retain this metal makes saliva lead concentration a potential indicator of current exposure.     

Novel tissue and cell type-specific gene/drug delivery system using surface engineered hepatitis B virus nano-particles

The hepatitis B virus (HBV) surface antigen (HBsAg) L particle is a hollow nano-scale particle. HBsAg L particles have many properties that make them useful for in vivo gene transfer vectors and drug delivery systems. Gene therapy so far has required the in vivo pinpoint delivery of genetic materials into the target organs and cells. Gene transfer by HBsAg L particles might be an attractive method, since their tropism is the same as that of HBV. The HBsAg L particles are able to deliver therapeutic payloads with high specificity to human hepatocytes. In addition, the specificity of L particle can be altered by displaying various cell-binding molecules on the surface. Our results indicate that the L particle is suitable for a cell- and tissue-specific gene/drug transfer vector. In this review, we discuss HBsAg L particles as a gene/drug transfer vector and its potential for the treatment of infectious diseases.     

Salivary gland scintigraphy in gastro-esophageal reflux disease

Gastro-esophageal reflux disease (GERD) is associated with a decreased salivary flow as well as gastric acid production. This study therefore aimed to investigate functional disorders of salivary glands in patients with GERD. Methods: Thirty-one consecutive patients with GERD underwent salivary gland scintigraphy. Results: If the results defined the optimal cutoff point for determining the decreased salivary secretion as 51 % in parotid glands and 36 % in submandibular glands, a decreased salivary secretion of right parotid gland, left parotid gland, right submandibular gland, and left submandibular gland was found in 39 %, 32 %, 36 %, and 58 %, respectively. Overall, salivary function disorder of at least one major salivary gland was found in 24 patients (78 %) with GERD. There was no difference in the incidence of impaired salivary function between GERD patients with and without erosive esophagitis. Salivary gland function was more frequently diminished than expected in GERD. We concluded that the presence of impaired salivary gland function was considered to be one of risk factors for developing GERD symptoms. Received 12 July 2006; accepted 10 September 2006     

Development of gene delivery system into skeletal muscles by bubble liposomes and ultrasound

Skeletal muscle is a promising target tissue for the gene therapy of both muscle and non-muscle disorders. Gene transfer into muscle tissue can produce a variety of physiologically active proteins and may ultimately be applied to treatment of many diseases. A variety of methods have been studied to transfer genes into skeletal muscle, including viral and non-viral vectors. Recently, we have developed the polyethyleneglycol (PEG)-modified liposomes entrapping echo-contrast gas known as ultrasound (US) imaging gas. We have called the liposomes "Bubble liposomes" (BLs). We have further demonstrated that US-mediated eruption of BLs loaded with naked plasmid-DNA is a feasible and efficient technique for gene delivery. In this study, to assess the feasibility and the effectiveness of BLs for the gene therapy of disorders, we tried to deliver therapeutic genes (anti-inflammatory cytokine; IL-10 or anti-angiogenic factor; hK1-5) into skeletal muscles of arthritis or tumor model mice by the gene delivery system with BLs and US exposure. As a result, their disease symptom was efficiently improved by the systemic secretion of therapeutic proteins. Thus, this US-mediated BLs technique for muscle gene transfer may provide an effective noninvasive method for arthritis or cancer gene therapy in clinical use. In addition, it may be applicable for the gene therapy of other non-muscle and muscle disorders.